CN109908126A - Cysteine is preparing the application in antimycotic biofilm drug - Google Patents
Cysteine is preparing the application in antimycotic biofilm drug Download PDFInfo
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- CN109908126A CN109908126A CN201910333244.4A CN201910333244A CN109908126A CN 109908126 A CN109908126 A CN 109908126A CN 201910333244 A CN201910333244 A CN 201910333244A CN 109908126 A CN109908126 A CN 109908126A
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Abstract
The present invention relates to technical field of medical chemistry, specifically application of the cysteine in the drug, medical instrument or medical material for preparing antimycotic biofilm, the fungi, which is candida albicans, cryptococcus, Aspergillus or chlosma, and the antimycotic biofilm is the growing multiplication for inhibiting fungal organism envelope/formation or for inhibiting/destroys established fungal organism envelope.The invention further relates to using cysteine in vitro non-therapeutic inhibit fungal organism envelope method.Cysteine used in the present invention can effectively inhibit the growth of fungal organism envelope, be applied to clinically antimycotic biofilm.
Description
Technical field
The present invention relates to technical field of medical chemistry, specifically, being that cysteine is preparing antimycotic biofilm medicine
Application in object, medical instrument or medical material.
Background technique
Cysteine (cysteine) is a kind of common sulfur-containing amino acid in organism, shown in structural formula such as formula (I),
Molecular formula C3H7NO2S, molecular weight 121.158.In recent years, the application of cysteine clinically is paid more and more attention, Ke Yiyou
The therapeutic radiation damage of effect ground and certain skin diseases.In addition, having anti-ageing since cysteine has antioxidation
Old effect.
Biofilm be microorganism contacted with debility object or living tissue surface and a kind of aggregation group for being formed, by it
The extracellular matrix itself generated is wrapped in the Tiny ecosystem of living bacterial cells formation, and the specific phenotypes with height drug resistance, are phases
The unique microbe survival mode of another kind for individually dispersing free state fungal cell.It is taken out from patient's body
The visible fungi of conduit or biological device surface tactophily in the form of biofilm.Biological quilt is formed to the fungi established in vitro
It is found when the model progress antifungal drug sensitivity experiments of film, bacterial strain shows as height drug resistance to common drug.Therefore, with
Biofilm form grows and shows the internal conduit of height drug resistance or biological device correlation fungal infection is controlled to clinic
Treatment brings great difficulty, forces clinician that must take out conduit or biology from patient's body while carrying out antifungal therapy
Device, to influence the prognosis of patient.However, in the prior art, the drug with antifungal activity does not often have antibiosis
Object causes it that cannot obtain expected effect when being used for the fungus therapy of growth in the form of biofilm by film activity.
Summary of the invention
The present inventor's in-depth study by long-term, it has unexpectedly been found that, cysteine has good antimycotic biology
By film activity, therefore pre- anti-fungal can be used very efficiently for and form biofilm, or inhibit and destroy established biology
Envelope.Based on above-mentioned discovery, inventor completes the present invention.
The purpose of the present invention is to provide cysteines to prepare antimycotic biofilm drug, medical instrument or medical material
New application in material.Another object of the present invention is to provide a kind of antimycotic biofilm drugs.Third mesh of the invention
Be a kind of method that external non-therapeutic inhibits fungal organism envelope is provided.
To achieve the above object, the present invention takes following technical scheme:
The first aspect of the present invention provides cysteine and is preparing the application in antimycotic biofilm drug.
Further, the cysteine is the compound with lower formula (I) structure:
Further, the drug can also include other with the antimycotic or antimycotic active drug of biofilm
Component.
Further, the drug is the growing multiplication (formation) for inhibiting fungal organism envelope, or for inhibiting
The drug of (or destruction) established fungal organism envelope.
Further, the fungi is one or more of candida albicans, cryptococcus, Aspergillus, chlosma
Combination.
Further, the antimycotic biofilm drug is liniment, emulsion, paste, aerosol, film.
Cysteine used in the present invention can effectively inhibit the growth of fungal organism envelope, and minimum effective application concentration is
5mg/L can apply to clinically antimycotic biofilm.
Further, when the antimycotic biofilm drug is anti-candida biofilm drug, cysteine
Effective concentration in the anti-candida biofilm drug is >=5 mg/litres or mg/kg.
Further, when the candida albicans biofilm is mature biofilm, cysteine is in drug
Effective concentration is >=10 mg/litres or mg/kg.
Further, when the antimycotic biofilm drug is anti-Cryptococcus neoformans biofilm drug, half Guang
Effective concentration of the propylhomoserin in the anti-Cryptococcus neoformans biofilm drug is >=20 mg/litres or mg/kg.
Further, when the cryptococcus biofilm is mature biofilm, cysteine is in drug
Effective concentration is >=40 mg/litres or mg/kg.
Further, when the antimycotic biofilm drug is anti-Aspergillus biofilm drug, cysteine
Effective concentration in the anti-Aspergillus biofilm drug is >=40 mg/litres or mg/kg.
Further, when the Aspergillus biofilm is mature biofilm, cysteine is in drug
Effective concentration is >=80 grams per liters or mg/kg.
Further, when the antimycotic biofilm drug is anti-chlosma biofilm drug, half Guang ammonia
Effective concentration of the acid in the anti-chlosma biofilm drug is >=20 mg/litres or mg/kg.
Further, when the chlosma biofilm is mature biofilm, cysteine is in drug
Effective concentration be >=40 mg/litres or mg/kg.
The second aspect of the present invention provides cysteine in the medical instrument or medical material for preparing antimycotic biofilm
In application.
Further, the medical instrument or medical material include: catheter (such as perfusion tube, catheter), oxygen therapy
Mask, nasal oxygen tube, pincers, dressing, shield traumatic material, absorbable hemostasia/adherence preventing material, surgical supplies (such as medical pincers, hook,
Or needle), implantation material etc..
Further, the medical instrument or medical material the preparation method comprises the following steps: sterilizing half Guang ammonia of effective quantity will be included
The drug coat of acid is in the medical instrument or medical material that on medical instrument or on medical material, form antimycotic biofilm.
Further, the minimum effective concentration of cysteine is 5mg/ in the drug of the sterilizing effective quantity cysteine
L。
Further, the preparation method of the medical instrument or medical material include: (1) provide a medical instrument or
Medical material；(2) drug coat comprising the effective quantity cysteine that sterilizes is anti-true in being formed on medical instrument or medical material
The medical instrument or medical material of bacterium biofilm.
Further, the medical instrument of the antimycotic biofilm or medical material are to inhibit fungal organism envelope
The medical instrument or medical material of growing multiplication (formation), or inhibit the Medical treatment device of (or destruction) established fungal organism envelope
Tool or medical material.
The third aspect of the present invention provides a kind of method of external non-therapeutic inhibition fungal organism envelope, including following
Step: in the occasion for needing to inhibit fungal organism envelope, administration of cysteine, to inhibit fungal organism envelope.
The fourth aspect of the present invention, provides a kind of antimycotic biofilm drug, and the active constituent of the drug is half
Cystine.
Further, the drug can also include other with the antimycotic or antimycotic active drug of biofilm
Component.
Further, the drug is the growing multiplication (formation) for inhibiting fungal organism envelope, or for inhibiting
The drug of (or destruction) established fungal organism envelope.
Further, the fungi is one or more of candida albicans, cryptococcus, Aspergillus, chlosma
Combination.
Further, the drug is liniment, emulsion, paste, aerosol, film.
Further, when the drug is anti-candida biofilm drug, cysteine is in the anti-beads
Effective concentration in bacterium biofilm drug is >=5 mg/litres or mg/kg.
Further, when the candida albicans biofilm is mature biofilm, cysteine is in drug
Effective concentration is >=10 mg/litres or mg/kg.
Further, when the drug is anti-Cryptococcus neoformans biofilm drug, cysteine resists in described
Effective concentration in Cryptococcus neoformans biofilm drug is >=20 mg/litres or mg/kg.
Further, when the cryptococcus biofilm is mature biofilm, cysteine is in drug
Effective concentration is >=40 mg/litres or mg/kg.
Further, when the drug is anti-Aspergillus biofilm drug, cysteine is in the anti-aspergillus
Effective concentration in bacterium biofilm drug is >=40 mg/litres or mg/kg.
Further, when the Aspergillus biofilm is mature biofilm, cysteine is in drug
Effective concentration is >=80 grams per liters or mg/kg.
Further, when the drug is anti-chlosma biofilm drug, cysteine is in the anti-horse
Drawing the effective concentration in color bacterium biofilm drug is >=20 mg/litres or mg/kg.
Further, when the chlosma biofilm is mature biofilm, cysteine is in drug
Effective concentration be >=40 mg/litres or mg/kg.
Bacterial strain
The present invention provides a kind of cysteines to prepare the new application in antimycotic biofilm drug.Wherein, described
Fungi can be the tunicate fungi of any tool, preferable example includes but is not limited to: candida albicans, cryptococcus, Aspergillus,
Chlosma etc..
Fungi of the invention can be located in vitro, such as the medical instrument inner wall for needing to sterilize；Biology can also be located at
In vivo, wherein the biology includes but is not limited to: people, cat, dog, mouse, rat, rabbit, cavy, monkey etc..
In the present invention, fungi used can obtain or cultivate by conventional method, can also be obtained by commercially available approach
It arrives, is such as separated from the body cell or tissue of the patient with corresponding disease, or from the mechanism or public affairs of any commercially available reference culture
Department, as ATCC is commercially available.It should be understood that bacterial strain sample used in an embodiment of the present invention is only exemplary example,
Any fungi for forming biofilm may be incorporated for test the compounds of this invention antimycotic biofilm activity, without by
The limitation of scope of embodiments.
Fungal organism envelope
Biofilm is germs collect group, is that microorganism irreversibly connects with debility object or living tissue surface
Touching, the extracellular matrix generated by itself are wrapped in the Tiny ecosystem that living bacterial cells are formed, the special table with height drug resistance
Type is the unique microbe survival mode of another kind for individually dispersion free state fungal cell.
The conduit taken out from patient's body or biological device surface, can be observed fungi and are sticked life in the form of biofilm
It is long.Discovery when forming the model progress antifungal drug sensitivity experiments of biofilm to the fungi established in vitro, bacterial strain is to normal
Height drug resistance is shown as with drug.Therefore, in the form of biofilm grow and show height drug resistance internal conduit or
Biological device correlation fungal infection brings great difficulty to clinical treatment, and clinician is forced to carry out the same of antifungal therapy
Shi Bixu takes out conduit or biological device from patient's body, to influence the prognosis of patient.In general, fungal organism envelope is to one
As antifungal drug height drug resistance, current most of substances for having antifungal activity do not have antimycotic biofilm activity simultaneously.
Antimycotic biofilm activity
Cysteine of the invention acts on fungi, can effectively act as the growing multiplication for inhibiting fungal organism envelope
Effect, or play the role of destroying established fungal organism envelope.
Fungi, to most antifungal drug resistances, has anti-yeast after the material surfaces such as polystyrene form biofilm
The most nonreactive fungal organism of the traditional Chinese medicine monomer compound of phase fungi activity is by film activity.
The experiment proved that cysteine has significant antimycotic biofilm activity in vitro, fungi can not only be inhibited
The adherency of biofilm, growing multiplication, and it is inhibited to fungal organism envelope has been formed.
In an embodiment of the present invention: effective concentration of the cysteine in anti-candida biofilm drug be >=
5 mg/litres or mg/kg；When the candida albicans biofilm is mature biofilm, the cysteine is in drug
Effective concentration be >=10 mg/litres or mg/kg.
Effective concentration of the cysteine in anti-cryptococcus biofilm drug is >=20 mg/litres or milligram/thousand
Gram；When the cryptococcus biofilm is mature biofilm, effective concentration of the cysteine in drug is >=40
Mg/litre or mg/kg.
Effective concentration of the cysteine in anti-Aspergillus biofilm drug is >=40 mg/litres or milligram/thousand
Gram；When the Aspergillus biofilm is mature biofilm, effective concentration of the cysteine in drug is >=80
Mg/litre or mg/kg.
Effective concentration of the cysteine in anti-chlosma biofilm drug be >=20 mg/litres or milligram/
Kilogram；When the chlosma biofilm is mature biofilm, effective concentration of the cysteine in drug is
>=40 mg/litres or mg/kg.
It should be understood that in an embodiment of the present invention, when only having write effective growth for inhibiting fungal organism envelope exactly, activity at
The minimum concentration divided.In conjunction with this field common knowledge it is found that when antimycotic active medicine component concentration increase when, it is described
Anti-mycotic efficiency can have some improvement.Therefore, any those skilled in the art passes through combination after having read this specification
The common knowledge of this field can inhibit fungal organism envelope to grow using the dosage for being higher than the minimum concentration, this
Kind, which changes, equally to be fallen within the scope of disclosure of the invention.
Antimycotic biofilm product
Antimycotic biofilm reactive compound provided by the invention may be directly applied to organism, it can also be used to prepare
Drug or pharmaceutical composition, and need to inhibit the medical instrument or medical material of fungi.
The drug or pharmaceutical composition can be any dosage form, as decoction, powder, pill, vina, pastille, jelly,
Agent, paste, sublimed preparation, microencapsulation, vein cream are pressed in plaster, medicinal tea, herbal leaven, cake agent, distillate medicinal water, stylus, line agent, medicated roll, nail agent, moxibustion
Agent, Liposomal formulation, aerosol, precursor medicine preparation, injection, mixture, peroral ampule agent, tablet, capsule, pill, cream
Agent, ointment, rubber plaster, film, sponginum, ionophore, cutaneous permeable agent etc.；Preferably liniment, emulsion, cream
The dosage forms such as agent, aerosol, film.
The drug or pharmaceutical composition can be directly used for organism, may be made as coating agent and is applied to various needs
In the medical instrument or medical material for inhibiting fungi, inhibit the activity of fungi on medical instrument or medical material as being coated on.
The medical instrument or medical material can be any required medical instrument or medical material for inhibiting fungi, such as surgical operation
Instrument, ophthalmic instruments, earnose-throat instruments, oral and dental instrument and equipement, utensil and surgical instrument, orthopaedic srugery (orthopaedics) hand
Art instrument, obstetrics and gynecology department, family planning operation instrument, injection puncture instrument, burn (shaping) section surgical instrument, common examination device
Tool, clinical inspection analytic instrument, medical chemical examination and infrastructure device utensil, extracorporal circulatory system and blood processing apparatus, implantation material, hand
Art room, emergency room, consulting room equipment and utensil, ward nursing equipment and utensil, sterilization and disinfection equipment and utensil, Medical cold
Treatment, low temperature, refrigerating equipment and utensil, dental material, medical material and dressing, medical sutures material and adhesive, doctor
With high molecular material and product, intervention equipment etc..
In another preferred example, the medical instrument or medical material include but is not limited to: catheter is (as being infused
Pipe, catheter), oxygen hood, nasal oxygen tube, pincers, dressing, shield traumatic material, absorbable hemostasia/adherence preventing material, surgical supplies
(such as medical pincers, hook or needle), implantation material.
The invention has the advantages that:
The present invention provides a kind of effective method for destroying fungi envelope, cysteine used in the present invention can be effectively
Inhibit the growth of fungi envelope, or inhibit mature biofilm, can apply to clinically antimycotic biofilm.
Detailed description of the invention
95392 biofilm formation of Fig. 1 cysteine antifungal effect.(A) cysteine antifungal biology
The proliferation function of envelope.(B) cysteine antifungal maturation biofilm acts on.Wherein, abscissa is that cysteine is dense
It spends (mg/L), ordinate is biofilm formation rate (%).* P < 0.05 is represented, * * represents P < 0.01.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.It should be understood that these embodiments
It is only illustrative of the invention and is not intended to limit the scope of the invention.The experiment side of actual conditions is not specified in the following example
Method, usually according to normal condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number
It calculates by weight.
Embodiment 1: the preparation of antimycotic biofilm agent
It takes 4g PVA, 0.7g sorb formic acid, 0.5g glycerol that 10ml water is added to mix, after infiltration swelling, is heated to 90 degrees Celsius
Make to dissolve, the cysteine for being ground into atomic powder is added, adds water 20ml, after mixing evenly, is stood in 45 degrees Celsius of heat preservations, remove degassing
Bubble.By glass plate be preheated to it is mutually synthermal after, film to thickness is about 0.15mm, in 70 degrees Celsius of dryings, demoulding.
Embodiment 2: the preparation of antimycotic biofilm emulsion
It takes distilled water 8ml to set in beaker, 4g rubber powder is added to be made into rubber cement, rubber cement is transferred in mortar, half Guang is added by several times again
Propylhomoserin, side edged, which is ground to colostrum, to be formed, and is then transferred to colostrum in measuring cup by several times with a small amount of distilled water, stirs lower dropwise addition Ni Bo
Golden ethyl ester alcoholic solution, finally plus distilled water is to full dose, stirs evenly to obtain the final product.
Embodiment 3: the preparation of antimycotic biofilm paste
It takes material medicine to wear into micro mist, is separately added into oily phase, water phase, emulsifier, preservative matrix by a certain percentage, heat
Stirring in water bath mixing is again stirring for that cysteine and matrix is made to be mixed thoroughly to obtain paste after being cooled to room temperature.
Embodiment 4: the preparation of antimycotic biofilm aerosol
Cysteine and water, ethyl alcohol are configured to solution by a certain percentage, pours into container at room temperature, fastens valve,
Then a certain amount of propellant is pressed by press, filtration pressure pours into container after propellant liquefaction.
Embodiment 5: the preparation of antimycotic biofilm medical instrument haemostatic clamp
Any medicaments uniformity of Example 1-4 is coated on hemostasis jaw surfaces, forms it into one layer of antimycotic biology
Envelope protective coating.
Embodiment 6: the effect to different candida albicans bacterial strain biofilms is applied alone in cysteine
Material and method
1, reagent
Cysteine: it is purchased from Sigma company.
XTT: it is purchased from Sigma company.
Menadione (menadione): it is purchased from Sigma company.
Cysteine is made into 10g/L solution with sterile water, and test medicine is saved in -20 DEG C.Before experiment, by medicine storage liquid
35 DEG C of incubators thawings are set in taking-up, are mixed well.
The PBS of XTT sterilizing is made into the saturated solution of 0.5mg/ml, with the filter filtration sterilization in 0.22 μm of aperture, first naphthalene
Quinone is made into 10mM solution with 100% acetone, and XTT, menadione are saved in -80 DEG C.Before experiment, the taking-up of medicine storage liquid is set 4 DEG C
Refrigerator, 35 DEG C of incubator gradients are melted, and are mixed well.
2, bacterial strain
Candida albicans (Candida albicans) is provided by Changhai hospital Mycology Lab, and it is not equal to pick up from Changhai hospital respectively
Room clinical sample, and through morphology and biochemical identification.
3, culture solution
RPMI 1640 culture medium: RPMI 1640 (Gibco BRL) 10g, NaHCO32.0g, morpholine propane sulfonic acid
(morpholinepropanesulfonic acid, MOPS) 34.5g adds tri-distilled water 900ml to dissolve, and 1N NaOH tune pH is extremely
7.0, it is settled to 1000ml, filtration sterilization, 4 DEG C of preservations.
Husky fort glucose agar medium (SDA): peptone 10g, glucose 40g, agar 18g add deionized water 900ml
2mg/ml chloramphenicol solution 50ml is added in dissolution, adjusts pH to 7.0, is settled to 1000ml, 4 DEG C of preservations after high pressure sterilization.
YPD culture solution: yeast extract 10g, peptone 20g, glucose 20g add tri-distilled water 900ml to dissolve, are settled to
1000ml, 4 DEG C of preservations after high pressure sterilization.
4, instrument
Water isolation type electro-heating standing-temperature cultivator (Shanghai leap medical apparatus and instruments factory)；
THZ-82A Desk type constant-temperatureoscillator oscillator (Shanghai leap medical apparatus and instruments factory)；
511 type enzyme micro-plate readers (Shanghai third analysis instrument factory).
5, cysteine antifungal biofilm proliferation experiment
White strain inoculated of reading is to 1ml YPD culture solution, and in 30 DEG C, 200rpm shaken cultivation activates 16h, is in fungi
Later period exponential phase of growth.Candida albicans is collected by centrifugation and washes away YPD culture medium with PBS buffer solution, it is then that candida albicans is thin
Born of the same parents are suspended in 1640 culture medium of RPMI and are counted using ultraviolet specrophotometer, and adjusting bacteria concentration is 1.0 × 106cells/
ml.Bacterium solution is inoculated into 96 orifice plate, 1~No. 11 hole, in 37 DEG C of insulating boxs after stationary culture 1.5h, washes and abandons supernatant and delayed with PBS
Rush solution cleaning twice.The fresh culture of the cysteine containing various concentration is added, in 37 DEG C of continuation stationary cultures to taking afterwards for 24 hours
Out, XTT reduction method measures biofilm metabolic activity.
6, cysteine antifungal maturation biofilm is tested
White strain inoculated of reading is to 1ml YPD culture solution, and in 30 DEG C, 200rpm shaken cultivation activates 16h, is in fungi
Later period exponential phase of growth.Candida albicans is collected by centrifugation and washes away YPD culture medium with PBS buffer solution, it is then that candida albicans is thin
Born of the same parents are suspended in 1640 culture medium of RPMI and are counted using ultraviolet specrophotometer, and adjusting bacteria concentration is 1.0 × 106cells/
ml.Bacterium solution is inoculated into 96 orifice plate, 1~No. 11 hole, in 37 DEG C of insulating boxs stationary culture for 24 hours after, wash and abandon supernatant and slow with PBS
Rush solution cleaning twice.The fresh culture of the cysteine containing various concentration is added, for 24 hours in 37 DEG C of continuation stationary cultures.
7, SMIC value determines
Candida albicans takes out biofilm plate, is gently washed with PBS 3 times after 37 DEG C of cultures for 24 hours, and 200ml is added in every hole
XTT/menadione is protected from light, 37 DEG C of incubation 2-3h, takes out biofilm plate, draws 100 μ l XTT/menadione to sterile
In 96 orifice plates, each hole OD value is surveyed in 492nm with enzyme micro-plate reader.With positive control boring ratio, decline 80% or more most with OD value
Drug concentration in low-concentration holes is SMIC80(drug concentration when biofilm growth 80% is suppressed).
8, experimental result
Fig. 1 inhibiting effect of the cysteine to candida albicans biofilm as the result is shown.When cysteine concentration be 5,
10, when 20,40,80mg/L, the proliferation function (Figure 1A) of good antifungal biofilm is shown, cysteine is worked as
Concentration when being 10,20,40,80,160mg/L, show good antifungal maturation biofilm effect (Figure 1B).
Table 1 is as the result is shown: cysteine antifungal biofilm is proliferated (biofilm formation) effect
SMIC80Value is 20mg/L, cysteine antifungal maturation biofilm (mature biofilm) proliferation function
SMIC80Value is 40mg/L.
The SMIC of 1. cysteine antifungal biofilm of table80Value
| Bacterial strain | SMIC80(mg/L)biofilm formation | SMIC80(mg/L)mature biofilm |
| Candida albicans 22644 | 20 | 40 |
| Candida albicans 95392 | 20 | 40 |
| Candida albicans 98211 | 20 | 40 |
| Candida albicans 20631 | 20 | 40 |
| Candida albicans 20372 | 20 | 40 |
Embodiment 7: the effect to different C. neoformans strain biofilms is applied alone in cysteine
Material and method
1, reagent
Cysteine: it is purchased from Sigma company.
XTT: it is purchased from Sigma company.
Menadione (menadione): it is purchased from Sigma company.
Cysteine is made into 10g/L solution with sterile water, and test medicine is saved in -20 DEG C.Before experiment, by medicine storage liquid
35 DEG C of incubators thawings are set in taking-up, are mixed well.
The PBS of XTT sterilizing is made into the saturated solution of 0.5mg/ml, with the filter filtration sterilization in 0.22 μm of aperture, first naphthalene
Quinone is made into 10mM solution with 100% acetone, and XTT, menadione are saved in -80 DEG C.Before experiment, the taking-up of medicine storage liquid is set 4 DEG C
Refrigerator, 35 DEG C of incubator gradients are melted, and are mixed well.
2, bacterial strain
Cryptococcus neoformans (Cryptococcus neoformans) is provided by Shanghai Changhai Hospital Mycology Lab, is picked up from respectively
Hospital's different department clinical sample, and through morphology and biochemical identification.
3, culture solution
DMEM high glucose medium: it is purchased from Invitrogen company.
Husky fort glucose agar medium (SDA): peptone 10g, glucose 40g, agar 18g add tri-distilled water 900ml molten
2mg/ml chloramphenicol solution 50ml is added in solution, adjusts pH to 7.0, is settled to 1000ml, 4 DEG C of preservations after high pressure sterilization.
YPD culture solution: yeast extract 10g, peptone 20g, glucose 20g add tri-distilled water 900ml to dissolve, are settled to
1000ml, 4 DEG C of preservations after high pressure sterilization.
4, instrument
Water isolation type electro-heating standing-temperature cultivator (Shanghai leap medical apparatus and instruments factory)；
THZ-82A Desk type constant-temperatureoscillator oscillator (Shanghai leap medical apparatus and instruments factory)；
511 type enzyme micro-plate readers (Shanghai third analysis instrument factory).
5, the anti-Cryptococcus neoformans biofilm proliferation experiment of cysteine
Cryptococcus neoformans is seeded to 1ml YPD culture solution, and in 30 DEG C, 200rpm shaken cultivation, activation for 24 hours, makes at fungi
In later period exponential phase of growth.Candida albicans is collected by centrifugation and washes away YPD culture medium with PBS buffer solution, then by Novel hidden ball
Bacterium cell suspension is counted in DMEM culture medium and using ultraviolet specrophotometer, and adjusting bacteria concentration is 1.0 × 107cells/
ml.Bacterium solution is inoculated into 96 orifice plate, 1~No. 11 hole, in 37 DEG C of insulating boxs after stationary culture 1.5h, washes and abandons supernatant and delayed with PBS
Rush solution cleaning twice.The fresh culture of the cysteine containing various concentration is added, in 37 DEG C of continuation stationary cultures to taking afterwards for 24 hours
Out, XTT reduction method measures biofilm metabolic activity.
6, the anti-Cryptococcus neoformans maturation biofilm experiment of cysteine
Cryptococcus neoformans is seeded to 1ml YPD culture solution, and in 30 DEG C, 200rpm shaken cultivation, activation for 24 hours, makes at fungi
In later period exponential phase of growth.Candida albicans is collected by centrifugation and washes away YPD culture medium with PBS buffer solution, then by Novel hidden ball
Bacterium cell suspension is counted in DMEM culture medium and using ultraviolet specrophotometer, and adjusting bacteria concentration is 1.0 × 107cells/
ml.Bacterium solution is inoculated into 96 orifice plate, 1~No. 11 hole, in 37 DEG C of insulating boxs stationary culture for 24 hours after, wash and abandon supernatant and slow with PBS
Rush solution cleaning twice.The fresh culture of the cysteine containing various concentration is added, for 24 hours in 37 DEG C of continuation stationary cultures.
7, SMIC value determines
Cryptococcus neoformans takes out biofilm plate, is gently washed with PBS 3 times after 37 DEG C of cultures for 24 hours, and 200ml is added in every hole
XTT/menadione is protected from light, 37 DEG C of incubation 2-3h, takes out biofilm plate, draws 100 μ l XTT/menadione to sterile
In 96 orifice plates, each hole OD value is surveyed in 492nm with enzyme micro-plate reader.With positive control boring ratio, decline 80% or more most with OD value
Drug concentration in low-concentration holes is SMIC80(drug concentration when biofilm growth 80% is suppressed).
8, experimental result
Table 2 is as the result is shown: anti-Cryptococcus neoformans biofilm proliferation (biofilm formation) effect of cysteine
SMIC80Value is 20mg/L, the anti-Cryptococcus neoformans maturation biofilm of cysteine (mature biofilm) proliferation function
SMIC80Value is 40mg/L.
The SMIC of the anti-Cryptococcus neoformans biofilm of 2. cysteine of table80Value
| Bacterial strain | SMIC80(mg/L)biofilm formation | SMIC80(mg/L)mature biofilm |
| Cryptococcus neoformans 423 | 20 | 40 |
| Cryptococcus neoformans 780 | 20 | 40 |
| Cryptococcus neoformans 551 | 20 | 40 |
| Cryptococcus neoformans 597 | 20 | 40 |
| Cryptococcus neoformans 216 | 20 | 40 |
Embodiment 8: the effect to different aspergillus fumigatus bacterial strain biofilms is applied alone in cysteine
Material and method
1, reagent
Cysteine: it is purchased from Sigma company.
XTT: it is purchased from Sigma company.
Menadione (menadione): it is purchased from Sigma company.
Cysteine is made into 10g/L solution with sterile water, and test medicine is saved in -20 DEG C.Before experiment, by medicine storage liquid
35 DEG C of incubators thawings are set in taking-up, are mixed well.
The PBS of XTT sterilizing is made into the saturated solution of 0.5mg/ml, with the filter filtration sterilization in 0.22 μm of aperture, first naphthalene
Quinone is made into 10mM solution with 100% acetone, and XTT, menadione are saved in -80 DEG C.Before experiment, the taking-up of medicine storage liquid is set 4 DEG C
Refrigerator, 35 DEG C of incubator gradients are melted, and are mixed well.
2, bacterial strain
Aspergillus fumigatus (Aspergillus fumigatus) is provided by Shanghai Changhai Hospital Mycology Lab, picks up from the doctor respectively
Institute's different department clinical sample, and through morphology and biochemical identification.
3, culture solution
RPMI 1640 culture medium: RPMI 1640 (Gibco BRL) 10g, NaHCO32.0g, morpholine propane sulfonic acid
(morpholinepropanesulfonic acid, MOPS) (Sigma) 34.5g adds tri-distilled water 900ml to dissolve, 1N NaOH tune
PH to 7.0 (25 DEG C) is settled to 1000ml, filtration sterilization, 4 DEG C of preservations.
Husky fort glucose agar medium (SDA): peptone 10g, glucose 40g, agar 18g add tri-distilled water 900ml molten
2mg/ml chloramphenicol solution 50ml is added in solution, adjusts pH to 7.0, is settled to 1000ml, 4 DEG C of preservations after high pressure sterilization.
YPD culture solution: yeast extract 10g, peptone 20g, glucose 20g add tri-distilled water 900ml to dissolve, are settled to
1000ml, 4 DEG C of preservations after high pressure sterilization.
4, instrument
Water isolation type electro-heating standing-temperature cultivator (Shanghai leap medical apparatus and instruments factory)；
THZ-82A Desk type constant-temperatureoscillator oscillator (Shanghai leap medical apparatus and instruments factory)；
511 type enzyme micro-plate readers (Shanghai third analysis instrument factory).
5, cysteine anti-aspergillus fumigatus bacterium biofilm proliferation experiment
Aspergillus fumigatus bacterium is seeded to the inclined-plane SDA, 35 DEG C, is cultivated one week.After this method activation twice, add appropriate RPMI
1640 culture medium blows and beats bacterium colony in the inclined-plane SDA, with suction pipe, is free on aspergillus fumigatus bacterium spore in RPMI 1640 culture medium, so
It is filtered by four layers of sterile gauze.Culture solution adds RPMI 1640 culture medium to adjust spore concentration after blood cell counting plate counts
To 105Bacterium solution is inoculated into 96 orifice plates by cells/ml, in 37 DEG C of insulating boxs after stationary culture 1.5h, is washed and is abandoned supernatant and use PBS
Buffer solution cleans twice.The fresh culture of the cysteine containing various concentration is added, in 37 DEG C of continuation stationary cultures to after for 24 hours
It takes out, XTT reduction method measures biofilm metabolic activity.
6, cysteine anti-aspergillus fumigatus bacterium maturation biofilm is tested
Aspergillus fumigatus bacterium is seeded to the inclined-plane SDA, 35 DEG C, is cultivated one week.After this method activation twice, add appropriate RPMI
1640 culture medium blows and beats bacterium colony in the inclined-plane SDA, with suction pipe, is free on aspergillus fumigatus bacterium spore in RPMI 1640 culture medium, so
It is filtered by four layers of sterile gauze.Culture solution adds RPMI 1640 culture medium to adjust spore concentration after blood cell counting plate counts
To 105Bacterium solution is inoculated into 96 orifice plates by cells/ml, in 37 DEG C of insulating boxs stationary culture for 24 hours after, wash to abandon and supernatant and use PBS
Buffer solution cleans twice.The fresh culture of the cysteine containing various concentration is added, in 37 DEG C of continuation stationary cultures to after for 24 hours
It takes out, XTT reduction method measures biofilm metabolic activity.
7, SMIC value determines
Aspergillus fumigatus takes out biofilm plate, is gently washed with PBS 3 times after 37 DEG C of cultures for 24 hours, and 200ml is added in every hole
XTT/menadione is protected from light, 37 DEG C of incubation 2-3h, takes out biofilm plate, draws 100 μ l XTT/menadione to sterile
In 96 orifice plates, each hole OD value is surveyed in 492nm with enzyme micro-plate reader.With positive control boring ratio, decline 80% or more most with OD value
Drug concentration in low-concentration holes is SMIC80(drug concentration when biofilm growth 80% is suppressed).
8, experimental result
Table 3 is as the result is shown: cysteine anti-aspergillus fumigatus bacterium bacterium biofilm is proliferated (biofilm formation) effect
SMIC80Value is 40mg/L, cysteine anti-aspergillus fumigatus bacterium maturation biofilm (mature biofilm) proliferation function
SMIC80Value is 80mg/L.
The SMIC of 3. cysteine anti-aspergillus fumigatus bacterium biofilm of table80Value
| Bacterial strain | SMIC80(mg/L)biofilm formation | SMIC80(mg/L)mature biofilm |
| Aspergillus fumigatus 638a | 40 | 80 |
| Aspergillus fumigatus 533a | 40 | 80 |
| Aspergillus fumigatus 965f | 40 | 80 |
| Aspergillus fumigatus 509d | 40 | 80 |
| Aspergillus fumigatus 505a | 40 | 80 |
Embodiment 9: the effect to different Malassezia furfur bacterial strain biofilms is applied alone in cysteine
Material and method
1, reagent
Cysteine: it is purchased from Sigma company.
XTT: it is purchased from Sigma company.
Menadione (menadione): it is purchased from Sigma company.
Cysteine is made into 10g/L solution with sterile water, and test medicine is saved in -20 DEG C.Before experiment, by medicine storage liquid
35 DEG C of incubators thawings are set in taking-up, are mixed well.
The PBS of XTT sterilizing is made into the saturated solution of 0.5mg/ml, with the filter filtration sterilization in 0.22 μm of aperture, first naphthalene
Quinone is made into 10mM solution with 100% acetone, and XTT, menadione are saved in -80 DEG C.Before experiment, the taking-up of medicine storage liquid is set 4 DEG C
Refrigerator, 35 DEG C of incubator gradients are melted, and are mixed well.
2, bacterial strain
Malassezia furfur (Malasezzia furfur) is provided by Shanghai Changhai Hospital Mycology Lab, picks up from the doctor respectively
Institute's different department clinical sample, and through morphology and biochemical identification.
3, culture solution
Chlosma culture solution: glucose 20g/L, bovine bile 4g/L, glycerol 1ml/L, glycerin monostearate 0.5g/L,
1640 10g/L of polysorbas20 0.4g/L, RPMI, filtration sterilization, 4 DEG C of preservations.
4, instrument
Water isolation type electro-heating standing-temperature cultivator (Shanghai leap medical apparatus and instruments factory)；
THZ-82A Desk type constant-temperatureoscillator oscillator (Shanghai leap medical apparatus and instruments factory)；
511 type enzyme micro-plate readers (Shanghai third analysis instrument factory).
5, the anti-Malassezia furfur biofilm proliferation experiment of cysteine
Twice by Malassezia furfur activation, chlosma fluid nutrient medium, adjustment spore concentration to 10 is added6cells/
Bacterium solution is inoculated into 96 orifice plates by ml, in 37 DEG C of insulating boxs after stationary culture 1.5h, is washed and is abandoned supernatant and clear with PBS buffer solution
It washes twice.The fresh culture of the cysteine containing various concentration is added, in 37 DEG C of continuation stationary cultures to taking out afterwards for 24 hours, XTT is also
Former method measures biofilm metabolic activity.
6, the anti-Malassezia furfur maturation biofilm experiment of cysteine
Twice by Malassezia furfur activation, chlosma fluid nutrient medium, adjustment spore concentration to 10 is added6cells/
Bacterium solution is inoculated into 96 orifice plates by ml, in 37 DEG C of insulating boxs stationary culture for 24 hours after, wash to abandon and supernatant and be cleaned with PBS buffer solution
Twice.The fresh culture of the cysteine containing various concentration is added, in 37 DEG C of continuation stationary cultures to taking out afterwards for 24 hours, XTT is restored
Method measures biofilm metabolic activity.
7, SMIC value determines
Malassezia furfur takes out biofilm plate, is gently washed with PBS 3 times after 37 DEG C of cultures for 24 hours, and every hole is added
200ml XTT/menadione, is protected from light, 37 DEG C of incubation 2-3h, takes out biofilm plate, draws 100 μ l XTT/menadione
To in sterile 96 orifice plate, each hole OD value is surveyed in 492nm with enzyme micro-plate reader.With positive control boring ratio, with OD value decline 80% with
On minimum concentration hole in drug concentration be SMIC80(drug concentration when biofilm growth 80% is suppressed).
8, experimental result
Table 4 is as the result is shown: anti-Malassezia furfur biofilm proliferation (biofilm formation) effect of cysteine
SMIC80Value is 40mg/L, the anti-Malassezia furfur maturation biofilm of cysteine (mature biofilm) proliferation function
SMIC80Value is 80mg/L.
The SMIC of the anti-Malassezia furfur biofilm of 4. cysteine of table80Value
It should be understood that in an embodiment of the present invention, when only having write effective growth for inhibiting fungal organism envelope exactly, activity at
The minimum concentration divided.In conjunction with this field common knowledge it is found that when antimycotic active medicine component concentration increase when, it is described
Anti-mycotic efficiency can have some improvement.Therefore, any those skilled in the art passes through combination after having read this specification
The common knowledge of this field can inhibit fungal organism envelope to grow using the dosage for being higher than the minimum concentration, this
Kind, which changes, equally to be fallen within the scope of disclosure of the invention.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (10)
1. cysteine is preparing the application in antimycotic biofilm drug.
2. application according to claim 1, which is characterized in that the antimycotic biofilm drug is liniment, cream
Agent, paste, aerosol, film.
3. application of the cysteine in the medical instrument or medical material for preparing antimycotic biofilm.
4. application according to claim 3, which is characterized in that the medical instrument or medical of the antimycotic biofilm
Material the preparation method comprises the following steps: by the drug coat comprising cysteine on medical instrument or on medical material, formed antimycotic
The medical instrument or medical material of biofilm.
5. application according to claim 1 to 4, which is characterized in that the fungi is candida albicans, cryptococcus, aspergillus
The combination of one or more of bacterium, chlosma.
6. application according to claim 1 to 4, which is characterized in that the antimycotic biofilm is to inhibit fungi
The established fungal organism envelope of growing multiplication/formation or inhibition/destruction of biofilm.
7. a kind of method that external non-therapeutic inhibits fungal organism envelope, comprising the following steps: needing to inhibit fungal organism
The occasion of envelope, administration of cysteine, to inhibit fungal organism envelope.
8. method according to claim 7, which is characterized in that the fungi be candida albicans, cryptococcus, Aspergillus,
The combination of one or more of chlosma.
9. a kind of antimycotic biofilm drug, which is characterized in that the active constituent of the drug is cysteine.
10. drug according to claim 9, which is characterized in that the drug further includes other with antimycotic or anti-
The active drug component of fungal organism envelope.
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